23-yne-vitamin d3 derivative

ABSTRACT

To provide a novel vitamin D 3  derivative useful as a therapeutic agent for osteoporosis. 
     Provided is a vitamin D 3  derivative represented by the following formula (1) or a medicinally acceptable solvate thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkylcarbonyloxyalkyl group with each alkyl having 1 to 6 carbon atoms, or an arylcarbonyloxyalkyl group with the aryl having 6 to 10 carbon atoms and the alkyl having 1 to 6 carbon atoms; R 2  represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms or, together with the other R 2  and the carbon atom to which they are bound to, may form a cyclic alkyl group having 3 to 6 carbon atoms; R 3  represents an alkyl group having 1 to 6 carbon atoms or, together with the other R 3  and the carbon atom to which they are bound to, may form a cyclic alkyl group having 3 to 6 carbon atoms; X represents an oxygen atom or a methylene group; and n represents an integer of 1 or 2.

TECHNICAL FIELD

The present invention relates to a vitamin D₃ derivative or amedicinally acceptable solvate thereof which is useful as a drug, to atherapeutic agent using the same, to a pharmaceutical compositioncomprising the same, and to a production intermediate thereof. Morespecifically, the present invention relates to a 23-yne-vitamin D₃derivative or a medicinally acceptable solvate thereof, to apharmaceutical composition comprising the same, to a therapeutic agentcomprising the same as an active ingredient for osteoporosis, malignanttumor psoriasis, hyperparathyroidism, inflammatory airway disease,rheumatoid arthritis, diabetes mellitus, hypertension, alopecia, acne,or dermatitis, and to a production intermediate thereof.

BACKGROUND ART

Activated vitamin D₃ derivatives regulate bone remodeling, consisting ofbone formation and bone resorption, and show bone a density-increasingeffect. Thus, they are being used as a valuable therapeutic agent forosteoporosis. However, these active vitamin D₃ derivatives,1α,25-dihydroxyvitamin D₃ for example, do not necessarily show asatisfactory amount of increase in the bone mineral density. When thedose is increased in order to increase the bone mineral density, thereoccurs an increase in a serum calcium value rather than further increasein the bone mineral density, causing elevation of the serum calciumvalue by 1 mg/dL or more, a value considered as one of the criteria forclinical undesirability. Thus, there are occasional cases where asufficient bone mineral density-increasing effect is not obtained(International Publication No. WO 01/62723). Therefore, there is anearnest desire for an activated vitamin D₃ derivative which exhibits astrong bone mineral density-increasing effect without increasing theserum calcium value. Heretofore, there have been synthesized a multitudeof vitamin D₃ derivatives in an effort to obtain such a derivative, butthere has not yet been found any derivative which has a satisfactoryprofile.

SUMMARY OF INVENTION

An object of the present invention is to provide a novel vitamin D₃derivative or a medicinally acceptable solvate thereof which exhibits adesired pharmacological effect isolated from the blood calciumincreasing effect.

Further, an object of the present invention is to provide a therapeuticagent for osteoporosis, malignant tumor, psoriasis, hyperparathyroidism,inflammatory airway disease, rheumatoid arthritis, diabetes mellitus,hypertension, alopecia, acne, or dermatitis, comprising the vitamin D₃derivative or a pharmaceutically acceptable solvate thereof as an activeingredient.

Furthermore, an object of the present invention is to provide apharmaceutical composition comprising the vitamin D₃ derivative or anmedicinally acceptable solvate thereof.

Still further, an object of the present invention is to provide anintermediate of the vitamin D₃ derivative suitable for producing thevitamin D₃ derivative or a medicinally acceptable solvate thereof.

The present inventors conducted diligent research in order to solve theabove-mentioned problems and, as a result, reached the followinginvention.

That is, the present invention is a vitamin D₃ derivative represented bythe following formula (1) or a medicinally acceptable solvate thereof.

wherein

R₁ represents a hydrogen atom, an alkyl group having 1 to 6 carbonatoms, an alkylcarbonyloxyalkyl group with each alkyl having 1 to 6carbon atoms, or an arylcarbonyloxyalkyl group with the aryl having 6 to10 carbon atoms and the alkyl having 1 to 6 carbon atoms; R₂ representsa hydrogen atom or an alkyl group having 1 to 6 carbon atoms or,together with the other R₂ and the carbon atom to which they are boundto, may form a cyclic alkyl group having 3 to 6 carbon atoms; R₃represents an alkyl group having 1 to 6 carbon atoms or, together withthe other R₃ and the carbon atom to which they are bound to, may form acyclic alkyl group having 3 to 6 carbon atoms; X represents an oxygenatom or a methylene group; and n represents an integer of 1 or 2.

Further, the present invention is a pharmaceutical compositioncomprising the vitamin D₃ derivative represented by the above formula(1) or a medicinally acceptable solvate thereof, and a pharmaceuticallyacceptable carrier.

Furthermore, the present invention is a therapeutic agent for one ormore diseases selected from the group consisting of osteoporosis,malignant tumor, psoriasis, hyperparathyroidism, inflammatory airwaydisease, rheumatoid arthritis, diabetes mellitus, hypertension,alopecia, acne, and dermatitis, comprising the vitamin D₃ derivativerepresented by the above formula (1) or a medicinally acceptable solvatethereof as an active ingredient.

Still further, the present invention is a production intermediate forthe vitamin D₃ derivative, the intermediate being represented by formula(2):

wherein R₂, X, and n are the same as in the above formula (1); R₄represents R₁ in the above formula (1), a methoxymethyl group, amethoxyethoxymethyl group, a tetrahydrofuranyl group, atetrahydropyranyl group, or a benzyloxy methyl group; and R₅ representsa protecting group for a hydroxyl group.

According to the present invention, there is provided a novel vitamin D₃derivative or a medicinally acceptable solvate thereof, which iseffective for treating various diseases represented by osteoporosis,malignant tumor, psoriasis, hyperparathyroidism, inflammatory airwaydisease, rheumatoid arthritis, diabetes mellitus, hypertension,alopecia, acne, dermatitis and the like. Further, the productionintermediate represented by the above formula (2) of the presentinvention is useful for producing the vitamin D₃ derivative and the likeof the present invention.

DESCRIPTION OF EMBODIMENTS

The terms used in the present invention are defined as follows.

The “alkyl group” means a linear, branched, or cyclic aliphatichydrocarbon group. The alkyl group having 1 to 6 carbon atomsspecifically include, for example, a methyl group, an ethyl group, apropyl group, an isopropyl group, a butyl group, an isobutyl group, at-butyl group, a pentyl group, an isopentyl group, a hexyl group, acyclopropyl group, a cyclopropylmethyl group, and a cyclohexyl group.

The “alkylcarbonyloxyalkyl group” specifically includes at-butylcarbonyloxymethyl group.

The “arylcarbonyloxyalkyl group” specifically includes aphenylcarbonyloxymethyl group.

In the above formula (1), R₁ represents a hydrogen atom, an alkyl grouphaving 1 to 6 carbon atoms, an alkylcarbonyloxyalkyl group with eachalkyl having 1 to 6 carbon atoms, or an arylcarbonyloxyalkyl group withthe aryl having 6 to 10 carbon atoms and the alkyl having 1 to 6 carbonatoms. Among these, preferable is a hydrogen atom, a methyl group, anethyl group, a propyl group, an isopropyl group, or a t-butyl group; andespecially preferable is a hydrogen atom or an isopropyl group. As thealkylcarbonyloxyalkyl group, preferable is a t-butylcarbonyloxymethylgroup. Further, preferable as the arylcarbonyloxyalkyl group is aphenylcarbonyloxyalkyl group.

In the above formula (1), R₂ represents a hydrogen atom or an alkylgroup having 1 to 6 carbon atoms or, together with the other R₂ and thecarbon atom to which they are bound to, may form a cyclic alkyl grouphaving 3 to 6 carbon atoms. Among these, R₂ is preferably a hydrogenatom or a methyl group; or when R₂, together with the other R₂ and thecarbon atom to which they are bound, forms a cycloalkyl group,preferable is a cyclopropyl group.

In the above formula (1), R₃ represents an alkyl group having 1 to 6carbon atoms or, together with the other R₃ and the carbon atom to whichthey are bound to, may form a cyclic alkyl group. As the alkyl grouphaving 1 to 6 carbon atoms, preferable are a methyl group and an ethylgroup. Further, when R₃, together with the other R₃ and the carbon atomto which they are bound, forms a cycloalkyl group, preferable is acyclopentyl group.

Further, in the above formula (1), X represents an oxygen atom or amethylene group.

Furthermore, in the above formula (1), n represents an integer of 1 or2, where especially preferably n is 1.

As preferred specific examples of the vitamin D₃ derivative representedby the formula (1) of the present invention, there may be mentioned thecompounds shown in the following table.

TABLE 1 Compound No. R₁ R₂ R₃ X n C-1 Hydrogen atom Hydrogen MethylOxygen 1 atom group atom C-2 Methyl group Hydrogen Methyl Oxygen 1 atomgroup atom C-3 Ethyl group Hydrogen Methyl Oxygen 1 atom group atom C-4Propyl group Hydrogen Methyl Oxygen 1 atom group atom C-5 Isopropylgroup Hydrogen Methyl Oxygen 1 atom group atom C-6 t-Butyl groupHydrogen Methyl Oxygen 1 atom group atom C-7 t-Butylcarbonyl- HydrogenMethyl Oxygen 1 oxymethyl group atom group atom C-8 Phenylcarbonyl-Hydrogen Methyl Oxygen 1 oxymethyl group atom group atom D-1 Hydrogenatom Hydrogen Methyl Methylene 1 atom group group D-2 Methyl groupHydrogen Methyl Methylene 1 atom group group D-3 Ethyl group HydrogenMethyl Methylene 1 atom group group D-4 Propyl group Hydrogen MethylMethylene 1 atom group group D-5 Isopropyl group Hydrogen MethylMethylene 1 atom group group D-6 t-Butyl group Hydrogen Methyl Methylene1 atom group group E-1 Hydrogen Cyclopropyl Methyl Oxygen 1 atom groupatom E-2 Methyl Methyl Methyl Oxygen 1 group group group atom F-1Hydrogen Hydrogen Ethyl Oxygen 1 atom atom group atom F-2 HydrogenHydrogen Cyclopentyl Oxygen 1 atom atom atom

If necessary, the vitamin D₃ derivative of the present invention can beconverted to a medicinally acceptable solvate. Such a solvent includeswater, methanol, ethanol, 1-propanol, 2-propanol, butanol, t-butanol,acetonitrile, acetone, methyl ethyl ketone, chloroform, ethyl acetate,diethyl ether, t-butyl methyl ether, benzene, toluene, DMF, DMSO, andthe like. Especially, there may be mentioned water, methanol, ethanol,1-propanol, 2-propanol, acetonitrile, acetone, methyl ethyl ketone, andethyl acetate as preferable solvents.

In addition, R₅ in the above formula (2) represents a protecting groupfor a hydroxyl group. The protecting group for a hydroxyl group includesa methoxymethyl group, an acyl group having 1 to 3 carbon atoms (thenumber of carbon atoms includes the carbonyl carbon), a trimethylsilylgroup, a triethylsilyl group, a t-butyldimethylsilyl group, at-butyldiphenylsilyl group, and the like. Among these, a triethylsilylgroup and t-butyldimethylsilyl group may be mentioned as preferableexamples.

Further, R₄ in the above formula (2) represents R₁ in the above formula(1) or represents a methoxymethyl group, a methoxyethoxymethyl group, atetrahydrofuranyl group, a tetrahydropyranyl group, or a benzyloxymethylgroup. Among these, preferable is a methyl group, an ethyl group, apropyl group, an isopropyl group; or a t-butyl group, at-butylcarbonyloxymethyl group, a phenylcarbonyloxyalkyl group, or abenzyloxymethyl group.

Furthermore, in the above formula (2), n represents an integer of 1 or2, wherein n is especially preferably 1.

Synthesis of the vitamin D₃ derivative represented by the above formula(1) may be performed by any method but may, for example, be carried outas described in the following Scheme 1. That is, after compound (2) andcompound (3) are subjected to a coupling reaction, the protecting groupof the hydroxyl group is removed, and, if necessary, the ester group ishydrolyzed to obtain the target material (1).

In the reaction formula above, R₁ to R₅ in the compound (1) and thecompound (2) are the same as in the above formula (1) and formula (2).Further, in the reaction formula above, R₃ in the compound (3) are thesame as R₃ in the above formula (1). Furthermore, OPG in the compound(3) represents a protected hydroxyl group. Specifically, the protectinggroup includes a trimethylsilyl group, a triethylsilyl group, and amethoxymethyl group.

In the above Scheme 1, when R₂ is a hydrogen atom, the compound (2) canbe synthesized from an ene-yne compound (4) according to the followingScheme 2, the ene-yne compound (4) being described, for example, in aliterature (Takayama, et al., “Vitamin D Analog in Cancer Prevention andTherapy,” Recent Results in Cancer Research, Vol. 164, Springer, pp.289-317, 2003 and the like). That is, by selectively removing theprotecting group (t-butyldimethylsilyl group; TBS group) of the primaryhydroxyl group of (4), there is obtained compound (5). Then, thehydroxyl group of (5) is oxidized to a carboxyl group, which issubsequently esterified to obtain the desired (2) (R₂=TBS).

Meanwhile, in the above Scheme 1, when R₃ is a methyl group, compound(3) can be synthesized as described in the following Scheme 3.

That is, the compound (3) can be obtained by bromomethylenation ofcompound (6), the latter compound being described in a literature (forexample, U.S. Pat. No. 4,804,502).

Further, among the vitamin D₃ derivatives represented by the aboveformula (1), a compound wherein R₂ is a hydrogen atom can also besynthesized according to the method shown in the following Scheme 4 inaddition to the above Scheme 1. That is, the compound (5) in Scheme 2 isprotected with a pivaloyl group to obtain compound (7), which issubjected to coupling with the compound (3) in Scheme 1 and deprotectionof the hydroxyl group at the terminal of a substituent attached to2-position of the A ring to obtain compound (8). The hydroxyl group ofthe compound obtained is oxidized to a carboxylic acid and, finally, allprotecting groups of the hydroxyl groups are removed to obtain thecompound (1) (R₂═H).

Further, in the above Scheme 1, when R₂ is substituted, for example,when R₂ in compound (2) is forming a cycloalkyl group together with theother R₂ and the carbon atom to which they are bound to, compound (10)is obtained by epoxidizing commercial4,6-O-benzylidene-α-D-methyl-glucopyranoside (9), which is used as thestarting material, and subsequently carrying out ring opening of theepoxide under basic conditions, in the same way as the ene-yne compound(4) described in the literature (Takayama, et al., “Vitamin D Analog inCancer Prevention and Therapy,” Recent Results in Cancer Research, Vol.164, Springer, pp. 289-317, 2003 and the like). After obtaining compound(11) by protection of the hydroxyl groups of compound (10), ring openingof the benzylidene ring and, further, reduction of 1-position of glucosewere carried out to obtain compound (12). Subsequently, an epoxide isformed from the diol portion to obtain compound (13), followed byreaction of the epoxide with acetylene to obtain compound (14) having atriple bond site introduced. By suitably protecting the hydroxyl groups,compound (15) can be obtained. By coupling of the compound (15) and theCD ring intermediate (3) described in Scheme 1 and selectivedeprotection, there is obtained compound (16). Further, by oxidation ofthe primary hydroxyl group to a carboxylic acid and subsequentdeprotection of the protected hydroxyl groups, there can be obtained thedesired compound (1).

A therapeutic agent for osteoporosis and the like, comprising thevitamin D₃ derivative or a medicinally acceptable solvate thereof of thepresent invention as an active ingredient, is prepared by using acarrier, a vehicle, and other additives used commonly in drugformulation. The carrier and vehicle for drug formulation may be eithersolid or liquid, and include, for example, lactose, magnesium stearate,starch, talc, gelatin, agar, gum arabic, olive oil, sesame oil, cacaobutter, ethylene glycol, and the like; and other commonly usedmaterials. The mode of administration may be either oral administrationby means of tablets, pills, capsules, granules, powder, fluids, and thelike; or parenteral administration by means of injections such asintravenous injection, intramuscular injection, and the like,suppositories, transdermal drugs, and the like.

In the therapeutic agent of the present invention, a therapeuticallyeffective amount of the active ingredient varies depending on the routeof administration, age and gender of the patient, and extent of thedisease. However, it is generally about 0.01 to 10 μg/day and the numberof doses is generally 1 to 3 times/day or 1 to 3 times/week. Thus, theformulation is preferably prepared to satisfy these conditions.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to Examples. However, it should be understood that the presentinvention is not deemed to be limited thereto. In addition,abbreviations used in the present invention are as follows:

TBS=t-butyldimethylsilyl group;TES=triethylsilyl group;TESCl=chlorotriethylsilane;TMS=trimethylsilyl group;TMSCl=chlorotrimethylsilane;Piv=pivaloyl group;PivCl=pivaloyl chloride;TBAF=tetrabutylammonium fluoride;CSA=(+/−)-camphor-10-sulfonic acid;PDC=pyridinium dichromate;TBSOTf=t-butyldimethylsilyl trifluoromethanesulfonate;DIBAL=dibutylaluminum hydride;

DMF=N,N-dimethylformamide;

THF=tetrahydrofuran;TsCl=p-toluenesulfonyl chloride; andTs=p-toluenesulfonyl.

Example 1 Production of(5Z,7E)-(1R,2S,3R,20R)-2-(2-carboxyethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound C-1)

(1) Compound A-1 (2.29 g, 4.11 mmol), known in the literature (forexample, Kittaka et al., J. Org. Chem., 69, 7463-7471 (2004)), wasdissolved in ethanol (20 ml), thereto was added(+/−)-camphor-10-sulfonic acid (954 mg, 4.11 mmol) under ice cooling,and the mixture was stirred at 0° C. for 1 hour. The reaction wasterminated by the addition of saturated aqueous sodium hydrogencarbonate and the mixture was diluted with ethyl acetate. This waswashed with water and saturated aqueous sodium chloride and the organiclayer was dried over anhydrous sodium sulfate. The organic layer wasconcentrated under reduced pressure and the residue obtained waspurified by silica gel column chromatography (n-hexane/ethylacetate=9/1) to obtain compound A-2 (1.64 g, yield 90%).

¹H-NMR (CDCl₃) δ: 5.96-5.88 (1H, m), 5.27-5.21 (2H, m), 4.29 (1H, dd,J=6.8, 3.9 Hz), 3.88-3.72 (5H, m), 3.45 (1H, dd, J=5.4, 4.1 Hz), 3.00(1H, t, J=6.0 Hz), 2.50-2.46 (1H, m), 2.38-2.33 (1H, m), 2.01 (1H, t,J=2.6 Hz), 1.85-1.68 (2H, m), 0.91 (9H, s), 0.91 (9H, s), 0.10 (9H, s),0.07 (3H, s).

(2) The compound A-2 (1.0 g, 2.26 mmol) obtained in (1) was dissolved inpyridine (10 ml) and, after the addition of pivaloyl chloride (0.69 mL,5.65 mmol) at 0° C., the reaction mixture was stirred at roomtemperature. Anhydrous methanol (3 mL) was added thereto and the mixturewas stirred at room temperature for further 30 minutes. Thereto wasadded toluene and the mixture was concentrated under reduced pressure.To the residue obtained was added ethyl acetate, the mixture was washedwith saturated aqueous sodium chloride, and the organic layer was driedover anhydrous magnesium sulfate. The organic layer was concentratedunder reduced pressure and the residue obtained was purified by silicagel column chromatography (n-hexane/ethyl acetate=9/1) to obtaincompound A-3 (1.072 g, yield 90%).

¹H-NMR (CDCl₃) δ: 5.95 (1H, ddd, J=17.0, 11.0, 6.0 Hz), 5.21 (1H, ddd,J=17, 2.0, 1.0 Hz), 5.14 (1H, ddd, J=11.0, 2.0, 1.0 Hz), 4.32-4.28 (1H,m), 4.18-4.10 (2H, m), 3.86 (1H, q, J=5.6 Hz), 3.81-3.74 (1H, m),3.68-3.60 (1H, m), 3.39 (1H, dd, J=5.4, 3.4 Hz), 2.49 (1H, dq, J=17.0,2.7 Hz), 2.35 (1H, dq, J=16.9, 2.8 Hz), 1.96 (1H, t, J=2.7 Hz), 1.87(2H, dt, J=14.0, 7.0 Hz), 1.19 (9H, s), 0.90 (9H, s), 0.89 (9H, s), 0.10(3H, s), 0.08 (3H, s), 0.07 (5H, s), 0.03 (3H, s).

(3) (Bromomethyl)triphenylphosphonium bromide (1.25 g, 2.87 mmol) wasdissolved in tetrahydrofuran (7 ml) and the solution was cooled to 0° C.under a nitrogen atmosphere. Hereto was added sodiumbis(trimethylsilyl)amide (1.0 M tetrahydrofuran solution, 2.90 mL, 2.87mmol) and the mixture was stirred under ice cooling for 30 minutes. Thereaction mixture was cooled to −78° C. and thereto was added a solutionof compound B-1 (200 Mg, 0.574 mmol) dissolved in tetrahydrofuran, (1.5mL) the compound being known in the literature (for example, Uskokovicet al., U.S. Pat. No. 4,804,502). After stirring at −78° C. for 1 hour,the mixture was stirred at 0° C. for further 1 hour. To the reactionmixture was added silica gel (2.5 g) and, after vigorous stirring atroom temperature for 10 minutes, the mixture was filtered throughcelite. The filtrate obtained was concentrated under reduced pressureand the residue was purified by silica gel column chromatography(n-hexane/ethyl acetate=9/1) to obtain compound B-2 (161 mg, yield 67%).

¹H-NMR (CDCl₃) δ: 5.65 (1H, s), 2.90-2.86 (1H, m), 2.28-1.24 (20H, m),1.08 (3H, d, J=6.3 Hz), 0.58 (3H, s), 0.18 (9H, s).

(4) The compound B-2 (1.2 g, 2.82 mmol) obtained in (3) was dissolved intetrahydrofuran (10 mL), thereto was added tetrabutylammonium fluoride(1 M tetrahydrofuran solution, 4.23 mL, 4.23 mmol), and the mixture wasstirred at 50° C. for 30 minutes. Thereto was added ethyl acetate, themixture was washed with water, and the organic layer was dried overanhydrous magnesium sulfate. The organic layer was concentrated underreduced pressure and the residue obtained was purified by silica gelchromatography (n-hexane/ethyl acetate=19/1). The purified material wasdissolved in anhydrous pyridine (10 mL) and the solution was cooled to0° C. under a nitrogen atmosphere. Hereto was added chlorotriethylsilane(0.944 mL, 5.70 mmol) and the mixture was warmed to room temperature andstirred for 2.5 hours. The reaction mixture was cooled to 0° C. and,after the addition of saturated aqueous ammonium chloride and water,extraction was performed with toluene. The organic layer was washed withsaturated aqueous sodium chloride and dried over anhydrous magnesiumsulfate. The organic layer was concentrated under reduced pressure andthe residue obtained was purified by silica gel chromatography(n-hexane/ethyl acetate=99/1) to obtain Compound B-3 (783 mg, yield88%).

¹H-NMR (CDCl₃) δ: 5.65 (1H, s), 2.92-2.85 (1H, m), 2.23 (1H, dd, J=16.5,3.4 Hz), 2.07-1.24 (19H, m), 1.08 (3H, d, J=6.6 Hz), 0.96 (9H, t, J=7.9Hz), 0.66 (6H, q, J=7.9 Hz), 0.57 (3H, s).

(5) The compound B-3 (783 mg, 1.67 mmol) obtained in (4) and thecompound A-3 (733 mg, 1.39 mmol) obtained in (2) were dissolved inanhydrous toluene/triethylamine (1/1, 11.1 mL), thereto was addedtetrakis(triphenylphosphine)palladium (289 mg, 0.25 mmol), and themixture was stirred under a nitrogen atmosphere at 105° C. for 2 hours.After cooling to room temperature, diamine silica gel (produced by FujiSilysia Chemical Ltd., 6 g) and n-hexane (20 mL) were added thereto andthe mixture was stirred at room temperature for 1 hour. Thereafter, themixture was filtered by using ethyl acetate, the filtrate obtained wasconcentrated under reduced pressure, and the residue was purified bysilica gel chromatography (n-hexane/ethyl acetate=100/0→95/5). Thepurified material obtained was dissolved in anhydrous tetrahydrofuran(5.5 mL) and anhydrous methanol (4.6 mL), thereto was added a methanolsolution of sodium methoxide (0.91 mL, 5.46 mmol), and the mixture wasrefluxed for 1 hour. Saturated aqueous ammonium chloride was added andthe mixture was concentrated under reduced pressure. To the residueobtained was added ethyl acetate, the mixture was washed with saturatedaqueous sodium chloride, and the organic layer was dried over anhydrousmagnesium sulfate. The organic layer was concentrated under reducedpressure and the residue obtained was purified by silica gelchromatography (n-hexane/ethyl acetate=100/0→50/50) to obtain compoundAB-1 (609 mg, yield 67%).

¹H-NMR (CDCl₃) δ: 6.18 (1H, d, J=11.2 Hz), 6.02 (1H, d, J=11.2 Hz), 5.30(1H, brs), 5.00 (1H, brs), 4.46 (1H, brs), 4.05 (1H, m), 3.88-3.69 (4H,m), 3.36 (1H, brs), 2.94 (1H, brs), 2.83-2.77 (1H, m), 2.62-2.56 (1H,m), 2.24 (1H, dd, J=16.5, 3.4 Hz), 2.10 (1H, dd, J=13.9, 4.4 Hz),2.06-1.21 (21H, m), 1.07 (3H, d, J=6.6 Hz), 0.96 (9H, t, J=7.9 Hz), 0.93(9H, s), 0.87 (9H, s), 0.67 (6H, q, J=7.9 Hz), 0.55 (3H, s), 0.10 (3H,s), 0.10 (3H, s), 0.08 (3H, s), 0.07 (3H, s).

(6) The compound AB-1 (427 mg, 0.514 mmol) obtained in (5) was dissolvedin anhydrous dichloromethane (5.2 mL) and the solution was cooled to 0°C. Thereafter, Dess-Martin reagent (523 mg, 1.23 mmol) was added and,after stirring under ice cooling for 2 hours, the mixture was warmed toroom temperature and stirred for 1 hour. Thereto were added saturatedaqueous sodium thiosulfate and saturated aqueous sodium hydrogencarbonate, and the mixture was extracted with dichloromethane. Theorganic layer was washed with saturated aqueous sodium chloride, driedover anhydrous sodium sulfate, and concentrated under reduced pressure.The residue obtained was dissolved in t-butanol (21 mL), thereto wereadded tetrahydrofuran (37 mL) and 2-methyl-2-butene (6.47 mL), and themixture was cooled with ice. An aqueous solution (7.3 mL) of sodiumhypochlorite (purity 80%, 580 mg, 5.14 mmol) and sodium dihydrogenphosphate dihydrate (400 mg, 2.57 mmol) was added and the mixture wasstirred under ice cooling for 45 minutes. Thereto were added saturatedaqueous sodium thiosulfate and saturated aqueous sodium hydrogencarbonate, and the mixture was extracted with ethyl acetate. The organiclayer was washed with saturated aqueous sodium chloride and dried overanhydrous sodium sulfate. The organic layer was concentrated underreduced pressure and the residue obtained was purified by silica gelchromatography (n-hexane/ethyl acetate=100/0→80/20) to obtain compoundAB-2 (341 mg, yield 78%).

¹H-NMR (CDCl₃) δ: 6.22 (1H, d, J=11.2 Hz), 6.00 (1H, d, J=11.2 Hz), 5.27(1H, brs), 4.99 (1H, brs), 4.45 (1H, brs), 4.07 (1H, m), 3.91 (2H, t,J=6.1 Hz), 3.36 (1H, brs), 2.84-2.77 (1H, m), 2.64 (2H, d, J=6.1, 1.5Hz), 2.60-2.53 (1H, m), 2.24 (1H, dd, J=16.5, 3.4 Hz), 2.13 (1H, dd,J=13.9, 5.4 Hz), 2.07-1.21 (19H, m), 1.07 (3H, d, J=6.3 Hz), 0.96 (9H,t, J=7.9 Hz), 0.90 (9H, s), 0.87 (9H, s), 0.67 (6H, q, J=7.9 Hz), 0.55(3H, s), 0.09 (3H, s), 0.09 (6H, s), 0.07 (3H, s).

(7) The compound AB-2 (140 mg, 0.165 mmol) obtained in (6) was dissolvedin acetone (1.65 mL) and the solution was cooled to 0° C. Thereafter, adiluted solution (1.65 mL) of hydrochloric acid (6N, 0.332 mL) inacetone was added and the mixture was stirred at room temperature for 4hours. Thereto was added n-hexane (3.3 mL) and the mixture was roughlypurified by silica gel chromatography (n-hexane/acetone=1/1) and thinlayer silica gel chromatography (n-hexane/acetone=4/5), and furtherpurified by reversed-phase HPLC (A=0.1% formic acid/1% methanol/4%acetonitrile/water; B=0.1% formic acid/5% water/19%methanol/acetonitrile; 0-2 min.: B=20%, 2-20 min.: B=20%→98%, 20-25min.: B=98%, 25-30 min.: B=20%) to obtain compound C-1 (34.9 mg, yield42%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=11.2 Hz), 6.00 (1H, d, J=11.2 Hz), 5.39(1H, d, J=1.9 Hz), 5.09 (1H, d, J=1.9 Hz), 4.50 (1H, d, J=2.9 Hz),4.36-3.58 (6H, m), 3.35 (1H, dd, J=8.1, 3.2 Hz), 2.86-2.79 (1H, m),2.72-2.57 (3H, m), 2.29-2.19 (2H, m), 2.04-1.20 (19H, m), 1.06 (3H, d,J=6.6 Hz), 0.54 (3H, s).

Example 2 Production of(5Z,7E)-(1R,2S,3R,20R)-2-(2-methoxycarbonylethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound C-2)

(1) The compound A-2 (1.45 g, 3.27 mmol) obtained in Example 1 (1) wasdissolved anhydrous dimethylformamide (15 mL), thereto was addedpyridinium dichromate (6.17 g, 16.4 mmol), and the mixture was stirredfor 12 hours. Water was added, the mixture was extracted with ethylacetate, and the organic layer obtained was dried over anhydrousmagnesium sulfate. The organic layer was concentrated under reducedpressure and the residue obtained was purified by silica gel columnchromatography (20% ethyl acetate/n-hexane) to obtain compound A-4 (0.82g, yield 55%).

¹H-NMR (CDCl₃) δ: 5.90 (1H, ddd, J=17.0, 6.0, 11.0 Hz), 5.30-5.20 (2H,m), 4.33 (1H, ddt, J=7.0, 3.0, 1.0 Hz), 3.96 (2H, td, J=6.0, 1.2 Hz),3.85-3.75 (1H, m), 3.55 (1H, dd, J=6.3, 3.7 Hz), 2.63 (2H, td, J=5.9,1.9 Hz), 2.50-2.32 (2H, m), 2.02 (1H, t, J=2.7 Hz), 0.91 (9H, s), 0.90(9H, s), 0.11 (3H, s), 0.10 (3H, s), 0.09 (3H, s), 0.08 (3H, s).

(2) The compound A-4 (0.82 g, 1.79 mmol) obtained in (1) was dissolvedin anhydrous methanol (8 mL), thereto was added concentrated sulfuricacid (74 μL, 1.5 mmol), and the mixture was stirred for 2.5 hours. Aftercooling to room temperature, saturated aqueous sodium hydrogen carbonatewas added thereto and the mixture was extracted with ethyl acetate. Theorganic layer obtained was dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was dissolved inanhydrous dichloromethane, thereto were added under ice cooling2,6-lutidine (1.01 mL, 9 mmol) and t-butyldimethylsilyltrifluoromethanesulfonate (1.65 mL, 7.2 mmol), and thereafter themixture was stirred at room temperature for 1 hour. Anhydrous methanol(1.5 mL) was added and the mixture was stirred at room temperature forfurther 10 minutes. Thereto was added n-hexane/ethyl acetate (9/1), themixture was washed with water, and the organic layer obtained was driedover anhydrous sodium sulfate. The organic layer was concentrated underreduced pressure and the residue was purified by silica gel columnchromatography (3% ethyl acetate/n-hexane) to obtain compound A-5 (683.4mg, yield 81%).

¹H-NMR (CDCl₃) δ: 5.94 (1H, ddd, J=10.0, 17.2, 6.5 Hz), 5.21 (1H, dt,J=17.3, 1.3 Hz), 5.14 (1H, dt, J=10.0, 1.3 Hz), 4.30 (1H, dd, J=6.8, 3.4Hz), 4.00-3.97 (1H, m), 3.88-3.82 (2H, m), 3.68 (3H, s), 3.40 (1H, dd,J=5.5, 3.5 Hz), 2.57 (2H, t, J=6.6 Hz), 2.48 (1H, dq, J=16.8, 2.7 Hz),2.35 (1H, dq, J=17.0, 2.8 Hz), 1.96 (1H, t, J=2.6 Hz), 0.90 (9H, s),0.89 (9H, s), 0.09 (3H, s), 0.08 (3H, s), 0.07 (3H, s), 0.03 (3H, s).

(3) The compound A-5 (47.0 mg, 0.1 mmol) obtained in (2) and thecompound B-2 (46.2 mg, 0.11 mmol) obtained in Example 1 (3) weredissolved in toluene/triethylamine (1/1, 2 mL), thereto was addedtetrakis(triphenylphosphine)palladium (12.5 mg, 0.0108 mmol), and themixture was stirred under a nitrogen atmosphere at 110° C. for 3 hours.The mixture was cooled to room temperature and concentrated underreduced pressure. The residue was roughly purified by thin layer silicagel chromatography (n-hexane/ethyl acetate=19/1). The crude purifiedmaterial obtained was dissolved in anhydrousdichloromethane/acetonitrile (1/1, 1 mL), thereto were added at 0° C.under a nitrogen atmosphere lithium tetrafluoroborate (78 mg, 0.8 mmol)and sulfuric acid (1 M acetonitrile solution, 0.08 mL, 0.08 mmol), andthe mixture was stirred for 30 minutes. Thereto was added saturatedaqueous sodium hydrogen carbonate and the mixture was extracted withethyl acetate. The organic layer obtained was washed with saturatedaqueous sodium chloride and dried over anhydrous sodium sulfate. Theorganic layer was concentrated under reduced pressure and the residueobtained was roughly purified by thin layer silica gel chromatography(n-hexane/ethyl acetate=1/2) and further purified by reversed-phase HPLC(A=95% water/acetonitrile; B=0.5% water/40% methanol/acetonitrile;B=75%) to obtain compound C-2 (6.8 mg, 13%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=11.2 Hz), 6.03 (1H, d, J=11.2 Hz), 5.40(1H, d, J=1.2 Hz), 5.09 (1H, d, J=2.2 Hz), 4.45 (1H, t, J=3.3 Hz),4.06-3.79 (3H, m), 3.73 (3H, s), 3.36 (1H, dd, J=7.7, 3.3 Hz), 2.85-2.60(7H, m), 2.24 (2H, dt, J=18.8, 5.9 Hz), 2.02-1.96 (3H, m), 1.89-1.82(2H, m), 1.72-1.54 (6H, m), 1.51 (6H, s), 1.47-1.24 (4H, m), 1.06 (3H,d, J=6.3 Hz), 0.54 (3H, s).

MS m/z 537.2 (M+23)+523.3 (M+18)+

Example 3 Production of(5Z,7E)-(1R,2S,3R,20R)-2-(2-propoxycarbonylethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound C-4)

(1) Using the compound A-4 (240 mg, 0.525 mmol) obtained in Example 2(1) as a raw material and replacing methanol with propanol, synthesiswas carried out in the same manner as in Example 2 (2) to obtaincompound A-6 (18.5 mg, yield 27%).

(2) Using the compound A-6 (40.5 mg, 0.081 mmol) obtained in (1) and thecompound B-2 (47 mg, 0.11 mmol) obtained in Example 1 (3) as rawmaterials, synthesis was carried out in the same manner as in Example 2(3) to obtain compound C-4 (6.8 mg, yield 15%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=11.2 Hz), 6.03 (1H, d, J=11.2 Hz), 5.39(1H, d, J=1.2 Hz), 5.09 (1H, d, J=2.2 Hz), 4.45 (1H, t, J=3.5 Hz), 4.08(2H, t, J=6.7 Hz), 4.06-3.95 (2H, m), 3.85-3.77 (1H, m), 3.36 (1H, dd,J=7.8, 3.2 Hz), 2.85-2.82 (1H, m), 2.79 (1H, d, J=4.1 Hz), 2.70-2.62(4H, m), 2.26-2.22 (2H, m), 2.03-1.98 (3H, m), 1.90-1.80 (3H, m),1.70-1.64 (7H, m), 1.58-1.53 (4H, m), 1.51 (6H, s), 1.48-1.45 (2H, m),1.40-1.20 (4H, m), 1.06 (3H, d, J=6.6 Hz), 0.94 (4H, t, J=7.4 Hz), 0.54(3H, s).

Example 4 Production of(5Z,7E)-(1R,2S,3R,20R)-2-(2-(1-methyl)ethoxycarbonylethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound C-5)

(1) Using the compound A-4 (240 mg, 0.525 mmol) obtained in Example 2(1) as a raw material and replacing methanol with isopropanol, synthesiswas carried out in the same manner as in Example 2 (2) to obtaincompound A-7 (157.4 mg, yield 60%).

(2) Using the compound A-7 (35 mg, 0.07 mmol) obtained in (1) and thecompound B-2 (44 mg, 0.11 mmol) obtained in Example 1 (3) as rawmaterials, synthesis was carried out in the same manner as in Example 2(3) to obtain compound C-5 (6.8 mg, yield 17%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=11.0 Hz), 6.03 (1H, d, J=11.5 Hz), 5.39(1H, d, J=1.5 Hz), 5.09-5.02 (2H, m), 4.45 (1H, t, J=3.5 Hz), 4.05-3.78(3H, m), 3.35 (1H, dd, J=7.7, 3.3 Hz), 2.85-2.58 (6H, m), 2.28-1.53(18H, m), 1.51 (6H, s), 1.46-1.30 (5H, m), 1.26 (3H, d, J=1.7 Hz), 1.24(3H, d, J=1.5 Hz), 1.06 (3H, d, J=6.3 Hz), 0.54 (3H, s).

Example 5 Production of(5Z,7E)-(1S,2S,3R,20R)-2-(2-carboxypropyl)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound D-1)

(1) Compound A-8 (0.72 g, 1.69 mmol), obtained from(3R,4R,5S)-3,5-bis[(t-butyldimethylsilyl)oxy]-4-[3-{(t-butyldimethylsilyl)oxy}propyl]oct-1-ene-7-yne,a compound known in the literature (for example, Saito, et al.,Tetrahedron, 60, 7951-7961 (2004)) in the same manner as in Example 1(1), was dissolved in dichloromethane (6.8 ml). Thereto were added at 0°C. triethylamine (0.47 mL, 3.37 mmol), trimethylamine hydrochloride (16mg, 0.169 mmol), and p-toluenesulfonyl chloride (0.48 g, 2.53 mmol) andthe mixture was stirred at room temperature for 1 hour. Saturatedaqueous sodium hydrogen carbonate was added thereto, the mixture wasextracted with ethyl acetate, and the organic layer was dried overanhydrous magnesium sulfate. The organic layer was concentrated underreduced pressure and the residue obtained was dissolved indimethylformamide (3 mL). Thereto were added sodium cyanide (199 mg,4.06 mmol) and sodium iodide (380 mg, 2.53 mmol), and the mixture wasstirred at 50° C. for 2 hours. Water was added and the mixture wasextracted with ethyl acetate. The organic layer was washed withsaturated aqueous sodium chloride, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure to obtain crudecompound A-9. This was dissolved in tetrahydrofuran (5 mL), thereto wasadded tetrabutylammonium fluoride (1 M tetrahydrofuran solution, 5.07mL, 5.07 mmol), and the mixture was stirred at 60° C. for 1 hour. Ethylacetate was added, the mixture was washed with water, and the organiclayer was dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The residue obtained was dissolved indimethylformamide (5 mL), thereto were added at 0° C. imidazole (460 mg,6.76 mmol), dimethylaminopyridine (21 mg, 0.169 mmol), andchlorotriethylsilane (0.851 mL, 5.07 mmol), and the mixture was stirredat 50° C. for 40 minutes. Saturated aqueous sodium hydrogen carbonatewas added thereto, the mixture was extracted with ethyl acetate, and theorganic layer was dried over anhydrous magnesium sulfate. The organiclayer was concentrated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography (1% ethylacetate/n-hexane→2% ethyl acetate/n-hexane→5% ethyl acetate/n-hexane→10%ethyl acetate/n-hexane) to obtain compound A-10 (531.3 mg, yield 72%).

¹H-NMR (CDCl₃) δ: 5.82 (1H, ddd, J=17.0, 10.0, 7.0 Hz), 5.17 (1H, dd,J=17.2, 1.1 Hz), 5.11 (1H, ddd, J=10.0, 2.0, 1.0 Hz), 4.00-3.95 (1H, m),2.42-2.37 (2H, m), 2.32 (2H, t, J=7.8 Hz), 1.97 (1H, t, J=2.6 Hz),1.85-1.65 (3H, m), 1.43-1.29 (2H, m), 1.26 (2H, t, J=7.2 Hz), 0.89 (19H,s), 0.09 (3H, s), 0.06 (3H, s), 0.06 (3H, s), 0.03 (3H, s).

(2) The compound A-10 (449.4 mg, 1.03 mmol) obtained in (1) wasdissolved in dichloromethane (5 mL), thereto was addeddiisobutylaluminum hydride (1 M toluene solution, 2.08 mL, 2.08 mmol)under cooling at −78° C., and the mixture was stirred at −78° C. for 50minutes. Anhydrous methanol (0.3 mL) was added and the mixture wasstirred at room temperature for 20 minutes. Further, saturated aqueoussodium potassium tartrate was added and the mixture was stirred for 10minutes. Ethyl acetate was added thereto, the mixture was washed withsaturated aqueous sodium chloride, and the organic layer was dried overanhydrous magnesium sulfate and concentrated under reduced pressure. Theresidue obtained was dissolved in tetrahydrofuran (6.9 mL), thereto wereadded t-butanol (6.9 mL) and 2-methyl-2-butene (4.5 g), and the mixturewas cooled with ice. An aqueous solution (6.9 mL) of sodium hypochlorite(931 mg, 10.3 mmol) and sodium dihydrogen phosphate (803 mg, 5.15 mmol)was added thereto and the mixture was stirred for 1 hour. This wasfollowed by the addition of saturated aqueous sodium thiosulfate and,further, by the addition of saturated aqueous sodium hydrogen carbonate,and the mixture was extracted with ethyl acetate. The organic layer waswashed with saturated aqueous sodium chloride, dried over anhydroussodium sulfate, and concentrated under reduced pressure. The residueobtained was purified by silica gel chromatography (n-hexane/ethylacetate=100/1→50/1→20/1→10/1→5/1→2/1) to obtain compound A-11 (220 mg,yield 47%).

¹H-NMR (CDCl₃) δ: 5.82 (1H, ddd, J=17.0, 10.0, 7.0 Hz), 5.17 (1H, dd,J=17.2, 1.1 Hz), 5.11 (1H, ddd, J=10.0, 2.0, 1.0 Hz), 4.00-3.95 (1H, m),2.42-2.37 (2H, m), 2.32 (2H, t, J=7.8 Hz), 1.97 (1H, t, J=2.6 Hz),1.85-1.65 (3H, m), 1.43-1.29 (2H, m), 1.26 (2H, t, J=7.2 Hz), 0.89 (19H,s), 0.09 (3H, s), 0.06 (3H, s), 0.06 (3H, s), 0.03 (3H, s).

(3) The compound A-11 (126.6 mg, 0.278 mmol) obtained in (2) wasdissolved in dimethylformamide (1.2 mL), thereto was added triethylamine(0.126 mL, 0.9 mmol) under cooling at 0° C., and the mixture was stirredfor 40 minutes. Thereto was added saturated aqueous sodium hydrogencarbonate and the mixture was extracted with ethyl acetate. The organiclayer was dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The residue obtained was purified by silica gelchromatography (n-hexane/ethyl acetate=95/5) to obtain compound A-12(126.5 mg, yield 79%).

¹H-NMR (CDCl₃) δ: 5.82 (1H, ddd, J=17.0, 10.0, 7.0 Hz), 5.17 (1H, dd,J=17.2, 1.1 Hz), 5.11 (1H, ddd, J=10.0, 2.0, 1.0 Hz), 4.00-3.95 (1H, m),2.42-2.37 (2H, m), 2.32 (2H, t, J=7.8 Hz), 1.97 (1H, t, J=2.6 Hz),1.85-1.65 (3H, m), 1.43-1.29 (2H, m), 1.26 (2H, t, J=7.2 Hz), 0.89 (19H,s), 0.09 (3H, s), 0.06 (3H, s), 0.06 (3H, s), 0.03 (3H, s).

(4) The compound A-12 (46 mg, 0.08 mmol) obtained in (3) above and thecompound B-2 (47 mg, 0.1 mmol) obtained in Example 1 (3) were dissolvedin toluene/triethylamine (1/1, 0.2 mL), thereto was addedtetrakis(triphenylphosphine)palladium (12 mg, 0.01 mmol), and themixture was stirred under a nitrogen atmosphere at 110° C. for 3 hours.The mixture was cooled to room temperature and, thereafter, concentratedunder reduced pressure. The residue was roughly purified by thin-layersilica gel column chromatography (n-hexane/ethyl acetate=19/1). Thecrude purified material obtained was dissolved in acetone, hydrochloricacid (6 N, 0.1 mL, 0.6 mmol) was added thereto, and the mixture wasstirred at 0° C. for 50 minutes. Further, hydrochloric acid (6 N, 0.2mL, 1.2 mmol) was added, and the mixture was stirred at room temperaturefor 40 minutes. Thereto was added saturated aqueous sodium hydrogencarbonate and the mixture was extracted with ethyl acetate. The organiclayer was dried over anhydrous magnesium sulfate, concentrated underreduced pressure, and the residue obtained was roughly purified by BondElut SI (produced by Varian, Inc.; n-hexane/ethyl acetate=1/2→ethylacetate→ethyl acetate/acetic acid=99/1). The crude purified material wasfurther purified by reversed-phase HPLC (A=95% water/acetonitrile;B=0.5% acetic acid/5% water/acetonitrile; B=65%) to obtain compound D-1(14.6 mg, 36%).

¹H-NMR (CDCl₃) δ: 6.40 (1H, d, J=11.5 Hz), 6.00 (1H, d, J=11.2 Hz), 5.27(1H, d, J=1.5 Hz), 4.99 (1H, d, J=2.0 Hz), 4.39 (1H, t, J=4.0 Hz),3.92-3.84 (1H, m), 2.86-2.79 (1H, m), 2.65 (1H, dd, J=13.3, 4.3 Hz),2.30-2.20 (4H, m), 2.05-1.96 (3H, m), 1.88 (2H, t, J=10.0 Hz), 1.81-1.64(8H, m), 1.56 (6H, dt, J=15.3, 4.5 Hz), 1.51 (6H, s), 1.49-1.46 (3H, m),1.45 (9H, s), 1.40-1.24 (5H, m), 1.06 (3H, d, J=6.6 Hz), 0.54 (3H, s),0.54 (3H, s).

Example 6 Production of(5Z,7E)-(1S,2S,3R,20R)-2-(2-(1,1-dimethyl)ethoxycarbonylpropyl)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound D-6)

(1) The compound A-9 (565 mg, 1.29 mmol) obtained in Example 5 (1) wasdissolved in dichloromethane, thereto was added diisobutylaluminumhydride (1 M toluene solution, 2 mL, 2 mmol) under cooling at −78° C.,and the mixture was stirred at −78° C. for 2 hours. Anhydrous methanol(1 mL) was added and the mixture was stirred at room temperature for 20minutes. Further, saturated aqueous sodium potassium tartrate was addedand the mixture was stirred for 10 minutes. Ethyl acetate was addedthereto, the mixture was washed with saturated aqueous sodium chloride,and the organic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue obtained was dissolvedin tetrahydrofuran (18.3 mL), thereto were added t-butanol (18.3 mL) and2-methyl-2-butene (6 mL), and the mixture was cooled with ice. Anaqueous solution (5 mL) of sodium hypochlorite (1.47 g, 13 mmol) andsodium dihydrogenphosphate (1.01 g, 6.5 mmol) was added thereto and themixture was stirred for 1 hour. To the mixture were added saturatedaqueous sodium thiosulfate and, further, saturated aqueous sodiumhydrogen carbonate, and the mixture was extracted with ethyl acetate.The organic layer was washed with saturated aqueous sodium chloride,dried over anhydrous sodium sulfate, and concentrated under reducedpressure. The residue obtained was purified by silica gel chromatography(n-hexane/ethyl acetate=9/1→7/1→5/1) to obtain compound A-13 (233.7 mg,yield 38%).

¹H-NMR (CDCl₃) δ: 5.82 (1H, ddd, J=17.0, 10.0, 7.0 Hz), 5.17 (1H, dd,J=17.2, 1.1 Hz), 5.11 (1H, ddd, J=10.0, 2.0, 1.0 Hz), 4.00-3.95 (1H, m),2.42-2.37 (2H, m), 2.32 (2H, t, J=7.8 Hz), 1.97 (1H, t, J=2.6 Hz),1.85-1.65 (3H, m), 1.43-1.29 (2H, m), 1.26 (2H, t, J=7.2 Hz), 0.89 (19H,s), 0.09 (3H, s), 0.06 (3H, s), 0.06 (3H, s), 0.03 (3H, s).

(2) To the compound A-13 (228.4 mg, 0.5 mmol) obtained in (1) were addedtoluene (5 mL) and N,N-dimethylformamide di-t-butyl acetal (1.1 mL, 4mmol), and the mixture was stirred at 80° C. for 1 hour. Thereto wasadded ethyl acetate, the mixture was washed with saturated aqueoussodium chloride, and the organic layer was dried over anhydrousmagnesium sulfate. The organic layer was concentrated under reducedpressure and the residue obtained was purified by silica gelchromatography (3% ethyl acetate/n-hexane) to obtain compound A-14(118.5 mg, yield 46%).

¹H-NMR (CDCl₃) δ: 5.83 (1H, ddd, J=17.0, 10.0, 7.0 Hz), 5.15 (1H, dq,J=17.2, 1.0 Hz), 5.10 (1H, dq, J=10.0, 1.0 Hz), 4.12 (1H, dd, J=8.0, 5.0Hz), 4.00 (1H, td, J=6.2, 3.8 Hz), 2.39 (2H, dd, J=6.1, 2.7 Hz), 2.17(2H, t, J=8.0 Hz), 1.79-1.63 (3H, m), 1.44 (9H, s), 1.40-1.20 (4H, m),0.89 (18H, s), 0.09 (3H, s), 0.06 (3H, s), 0.05 (3H, s), 0.03 (3H, s).

(3) The compound A-14 (59.6 mg, 0.12 mmol) obtained in (2) and thecompound B-2 (60 mg, 0.14 mmol) obtained in Example 1 (3) were dissolvedin toluene/triethylamine (1/1, 2 mL), thereto was addedtetrakis(triphenylphosphine)palladium (17 mg, 0.0147 mmol), and themixture was stirred under a nitrogen atmosphere at 110° C. for 3.5hours. The mixture was cooled to room temperature and concentrated underreduced pressure. The residue was roughly purified by thin-layer silicagel chromatography (n-hexane/ethyl acetate=19/1). The crude purifiedmaterial obtained was dissolved in tetrahydrofuran, tetrabutylammoniumfluoride (1 M tetrahydrofuran solution, 0.84 mL, 0.84 mmol) was addedthereto, and the mixture was stirred at 60° C. for 2 hours. Ethylacetate was added, the mixture was washed with water, and the organiclayer was dried over anhydrous magnesium sulfate. The organic layer wasconcentrated under reduced pressure and the residue obtained was roughlypurified by thin-layer silica gel chromatography (n-hexane/ethylacetate=1/1) and further purified by reversed-phase HPLC (A=95%water/acetonitrile; B=0.5% water/40% methanol/acetonitrile; B=85%) toobtain compound D-6 (5.0 mg, yield 7%).

¹H-NMR (CDCl₃) δ: 6.40 (1H, d, J=11.5 Hz), 6.00 (1H, d, J=11.2 Hz), 5.27(1H, d, J=1.5 Hz), 4.99 (1H, d, J=2.0 Hz), 4.39 (1H, t, J=4.0 Hz),3.92-3.84 (1H, m), 2.86-2.79 (1H, m), 2.65 (1H, dd, J=13.3, 4.3 Hz),2.30-2.20 (4H, m), 2.05-1.96 (3H, m), 1.88 (2H, t, J=10.0 Hz), 1.81-1.64(8H, m), 1.56 (6H, dt, J=15.3, 4.5 Hz), 1.51 (6H, s), 1.49-1.46 (3H, m),1.45 (9H, s), 1.40-1.24 (5H, m), 1.06 (3H, d, J=6.6 Hz), 0.54 (3H, s),0.54 (3H, s).

Example 7 Production of(5Z,7E)-(1R,2S,3R,20R)-2-((t-butylcarbonyloxy)methoxycarbonylethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound C-7)

(1) The Compound A-4 (164.3 mg, 0.360 mmol) obtained in Example 2 (1)was dissolved in anhydrous N,N-dimethylformamide (1.2 mL) and thesolution was cooled to 0° C. Triethylamine (0.15 mL, 1.08 mmol) andpivaloyloxymethyl chloride (0.104 mL, 0.719 mmol) were added thereto andthe mixture was stirred at room temperature for 1 hour. After 1 hour,sodium iodide (150 mg, 1.008 mmol) and potassium carbonate (140 mg,1.008 mmol) were added, and the mixture was stirred under heating at 50°C. for further 30 minutes. The reaction mixture was cooled to roomtemperature and, after dilution with water, the resulting mixtureextracted with ethyl acetate. The organic layer was washed withsaturated aqueous sodium chloride, dried over anhydrous magnesiumsulfate, and thereafter concentrated under reduced pressure. The residueobtained was purified by silica gel column chromatography(n-hexane/ethyl acetate=5/1) to obtain compound A-15 (158.0 mg, yield77%).

¹H-NMR (CDCl₃) δ: 5.98-5.90 (1H, m), 5.76 (2H, s), 5.21 (1H, dt,J=17.32, 1.46 Hz), 5.14 (1H, dt, J=10.37, 1.10 Hz), 4.30 (1H, dd,J=8.00, 3.00 Hz), 4.02-3.82 (3H, m), 3.42 (1H, dd, J=5.61, 3.41 Hz),2.62 (2H, t, J=6.71 Hz), 2.47 (1H, ddd, J=16.83, 2.68, 5.50 Hz), 2.34(1H, ddd, J=16.83, 2.76, 5.50 Hz), 1.96 (1H, t, J=2.68 Hz), 1.21 (9H,s), 0.90 (9H, s), 0.89 (9H, s), 0.09 (3H, s), 0.08 (3H, s), 0.07 (3H,s), 0.03 (3H, s).

(2) Using the compound A-15 (40 mg, 0.07 mmol) obtained in (1) and thecompound B-2 (36 mg, 0.085 mmol) obtained in Example 1 (3) as rawmaterials, synthesis was carried out in the same manner as in Example 2(3) to obtain compound C-7 (7.8 mg, yield 18%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=11.47 Hz), 6.02 (1H, d, J=11.22 Hz),5.81-5.76 (2H, m), 5.39 (1H, d, J=1.46 Hz), 5.09 (1H, d, J=2.20 Hz),4.44 (1H, s), 4.04-3.95 (2H, m), 3.85-3.80 (1H, m), 3.36 (1H, dd,J=7.56, 3.17 Hz), 2.85-2.57 (6H, m), 2.28-1.81 (8H, m), 1.59-1.24 (16H,m), 1.23 (9H, s), 1.06 (3H, d, J=6.59 Hz), 0.54 (3H, s).

Example 8 Production of(5Z,7E)-(1R,2S,3R,20R)-2-((phenylcarbonyloxy)methoxycarbonylethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound C-8)

Using the compound A-4 (175 mg, 0.383 mmol) obtained in Example 2 (1) asa raw material and replacing pivaloyloxymethyl chloride withbenzoyloxymethyl chloride, synthesis was carried out in the same manneras in Example 7 (1) to obtain compound A-16. Thereafter, using thecompound A-16 (41.3 mg, 0.07 mmol) and the compound B-2 (34 mg, 0.08mmol) obtained in Example 1 (3) as starting materials, synthesis wascarried out in the same manner as in Example 7 (2) to obtain compoundC-8 (4.9 mg, yield 11%).

¹H-NMR (CDCl₃) δ: 8.09-8.07 (2H, m), 7.62-7.44 (3H, m), 6.41 (1H, d,J=10.98 Hz), 6.05-6.01 (3H, m), 5.38 (1H, d, J=1.46 Hz), 5.07 (1H, d,J=1.95 Hz), 4.44 (1H, d, J=2.93 Hz), 4.05-3.97 (2H, m), 3.87-3.82 (1H,m), 3.36 (1H, dd, J=7.56, 3.17 Hz), 2.85-2.64 (4H, m), 2.32-2.18 (2H,m), 2.05-1.53 (9H, m), 1.49-1.24 (4H, m), 1.06 (3H, d, J=6.34 Hz), 0.55(3H, s).

Example 9 Production of(5Z,7E)-(1R,2S,3R,20R)-2-((2-carboxy-2,2-ethano)ethoxy)-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (Compound E-1)

(1) The compound A-17 (6.03 g, 22.8 mmol), described in the literature(for example, Kittaka et al., J. Org. Chem., 69, 7463-7471 (2004)), wasdissolved in N-methylpyrrolidone (60 mL), thereto was added potassiumt-butoxide (11.88 g, 114 mmol), and the mixture was stirred underheating at 130° C. for 4 hours. The reaction mixture was cooled to roomtemperature, water (240 mL) and then DIAION HP-20SS (produced byMitsubishi Chemical Corporation, 30 g (dry weight)) were added thereto,and the mixture was stirred at room temperature overnight. The reactionmixture was filtered, the solid material was washed with saturatedaqueous ammonium chloride (100 mL) and water (200 mL), and extractedwith acetone (500 mL). The extraction solution was concentrated underreduced pressure and diluted with ethyl acetate. The mixture was washedwith saturated aqueous sodium chloride and the organic layer was driedover anhydrous magnesium sulfate. The organic layer was concentratedunder reduced pressure and the residue obtained was purified by silicagel column chromatography (n-hexane/ethyl acetate=1/4) to obtaincompound A-18 (1.78 g, yield 21%).

¹H NMR (CDCl₃) δ: 7.51-7.36 (5H, m), 5.54 (1H, s), 4.61 (1H, s),4.40-4.29 (2H, m), 4.08 (1H, t, J=4.27 Hz), 4.01 (1H, dd, J=9.27, 2.68Hz), 3.93 (1H, br s), 3.83-3.75 (3H, m), 3.60-3.50 (3H, m), 3.41 (3H,s), 0.59-0.41 (3H, m).

(2) The compound A-18 (2.97 g, 8.10 mmol) obtained in (1) was dissolvedin anhydrous pyridine (30 mL) and the solution was cooled to 0° C.Thereto was added pivaloyl chloride (1.15 mL, 9.32 mmol) and the mixturewas stirred at the same temperature for 1 hour. Anhydrous methanol (3mL) was added thereto, and the mixture was stirred at room temperaturefor 5 minutes and concentrated under reduced pressure. The residue wasdissolved in toluene, the solution was washed with saturated aqueoussodium chloride, and, thereafter, the organic layer was dried overanhydrous magnesium sulfate. The organic layer was concentrated underreduced pressure and dried. This crude material was dissolved inanhydrous dichloromethane (20 mL), the solution was cooled to 0° C., andafter the addition of 2,6-lutidine (1.3 mL, 11.6 mmol) andt-butyldimethylsilyl trifluoromethanesulfonate (2.14 mL, 9.32 mmol)thereto, the mixture was stirred at room temperature for 1 hour.Anhydrous methanol (5 mL) was added and the mixture was concentratedunder reduced pressure. The residue was dissolved in toluene, thesolution was washed with water, and the organic layer was dried overanhydrous sodium sulfate. The solution was concentrated under reducedpressure and the residue obtained was purified by silica gel columnchromatography (5% ethyl acetate/n-hexane→10% ethyl acetate/n-hexane) toobtain compound A-19 (3.19 g, yield 69%).

¹H NMR (CDCl₃) δ: 7.49-7.34 (5H, m), 5.56 (1H, s), 4.45 (1H, s),4.29-4.25 (2H, m), 4.18 (1H, d, J=11.22 Hz), 3.98-3.92 (3H, m), 3.75(1H, t, J=12.08 Hz), 3.65 (1H, t, J=2.68 Hz), 3.56 (2H, dd, J=29.76,9.51 Hz), 3.35 (3H, s), 1.19 (9H, s), 0.91 (9H, s), 0.61-0.51 (4H, m),0.10 (3H, s), 0.10 (3H, s).

(3) The compound A-19 (3.17 g, 5.61 mmol) obtained in (2) was dissolvedin cyclohexane (63 mL), thereto were added barium carbonate (775 mg,3.92 mmol), benzoyl peroxide (136 mg, 0.56 mmol), and N-bromosuccinimide(1.21 g, 6.73 mmol), and the mixture was heated under reflux for 1 hour.After cooling, the mixture was filtered through celite, and the organiclayer was washed in the order of saturated aqueous sodium hydrogencarbonate and saturated aqueous sodium chloride. Thereafter, the organiclayer was dried over anhydrous magnesium sulfate and concentrated underreduced pressure to obtain a crude material (4.0 g). This crude materialwas dissolved in a mixed solvent of 1-propanol (36 mL) and water (4 mL),thereto were added activated zinc (7.38 g, 112.2 mmol) and sodiumcyanoborohydride (1.42 g, 22.4 mmol), and the mixture was heated underreflux for 1 hour. After cooling, the mixture was filtered throughcelite, the solid was washed with 1-propanol, and thereafter the liquidwas concentrated under reduced pressure. The residue obtained wasdiluted with ethyl acetate, washed with saturated aqueous sodiumchloride, and the organic layer was dried over anhydrous magnesiumsulfate. The organic layer was concentrated under reduced pressure andthe residue obtained was purified by silica gel column chromatography(hexane/ethyl acetate=90/10→80/20) to obtain compound A-20 (1.50 g,yield 50%).

¹H NMR (CDCl₃) δ: 8.05-8.02 (2H, m), 7.59-7.43 (3H, m), 6.11 (1H, ddd,J=11.00, 17.32, 6.00 Hz), 5.78-5.75 (1H, m), 5.41 (1H, dt, J=17.32, 1.34Hz), 5.30 (1H, dt, J=10.49, 1.22 Hz), 4.17 (1H, d, J=11.47 Hz),3.96-3.93 (2H, m), 3.81 (1H, dd, J=11.47, 5.12 Hz), 3.73-3.68 (2H, m),3.64 (1H, d, J=9.76 Hz), 3.50 (1H, d, J=9.76 Hz), 1.18 (9H, s), 0.90(9H, s), 0.55 (4H, t, J=1.95 Hz), 0.09 (3H, s), 0.07 (3H, s).

(4) The compound A-20 (2.41 g, 4.5 mmol) obtained in (3) was dissolvedin acetonitrile (25 mL), thereto were added triethylamine (1.26 mL, 9mmol), trimethylamine hydrochloride (86 mg, 0.9 mmol), andp-toluenesulfonyl chloride (1.30 g, 6.8 mmol) in this order, and themixture was stirred at room temperature for 1 hour. Saturated aqueoussodium hydrogen carbonate was added thereto and the mixture wasconcentrated under reduced pressure. The residue was diluted with ethylacetate and the mixture was washed with saturated aqueous sodiumchloride. The organic layer was dried over anhydrous magnesium sulfateand concentrated under reduced pressure. The crude material (3.31 g) wasdissolved in tetrahydrofuran (18 mL), thereto was addedtetrabutylammonium fluoride (1 M tetrahydrofuran solution, 13.5 mL, 13.5mmol), and the mixture was heated under reflux for 1.5 hours. Aftercooling, the mixture was concentrated under reduced pressure and theresidue was diluted with toluene. The mixture was washed with saturatedaqueous sodium chloride, and the organic layer was dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The residueobtained was purified by silica gel column chromatography (hexane/ethylacetate=90/10) to obtain compound A-21 (851 mg, yield 47%).

¹H NMR (CDCl₃) δ: 8.06-8.02 (2H, m), 7.61-7.44 (3H, m), 6.10-6.01 (1H,m), 5.67-5.64 (1H, m), 5.42 (1H, dt, J=17.24, 1.34 Hz), 5.32 (1H, dt,J=10.57, 1.22 Hz), 4.04 (2H, dd, J=27.32, 11.22 Hz), 3.65 (1H, d,J=10.24 Hz), 3.53 (1H, d, J=10.24 Hz), 3.17 (1H, dd, J=7.32, 5.37 Hz),3.10-3.06 (1H, m), 2.75 (1H, t, J=4.39 Hz), 2.60 (1H, dd, J=4.88, 2.93Hz), 1.19 (9H, s), 0.55 (4H, s).

(5) A tetrahydrofuran solution (3 mL) of trimethylsilylacetylene (1.62mL, 11.5 mmol) was placed under a nitrogen atmosphere and the solutionwas cooled with dry ice-acetone. Hereto was added a hexane solution ofn-butyllithium (2.64 M, 3.97 mL, 10.5 mmol) and the mixture was stirredfor 45 minutes. To this mixture were added a tetrahydrofuran solution (6mL) of the compound A-21 (846 mg, 2.1 mmol) obtained in (4) andtrifluoroborane-diethyl ether complex (0.343 mL, 2.73 mmol), and themixture was stirred for 2 hours under dry ice-acetone cooling andfurther stirred for 1 hour at 0° C. Saturated aqueous ammonium chloridewas added thereto, and the mixture was returned to room temperature anddiluted with ethyl acetate. The solution was washed successively withsaturated aqueous sodium hydrogen carbonate and saturated aqueous sodiumchloride, and the organic layer was dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The residue obtainedwas dissolved in anhydrous methanol (10 mL), sodium methoxide (870 mg,6.3 mmol) was added thereto, and the mixture was stirred under heatingat 50° C. for 1 hour. After cooling, the mixture was concentrated underreduced pressure. The residue was diluted with ethyl acetate, thereafterthe mixture was washed with saturated aqueous sodium chloride, and theorganic layer was dried over anhydrous magnesium sulfate. The organiclayer was concentrated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography (hexane/ethylacetate=60/40→50/50→35/65) to obtain Compound A-22 (311.5 mg, yield62%).

¹H NMR (CDCl₃) δ: 5.57 (1H, ddd, J=17.00, 11.00, 6.00 Hz), 4.88 (1H, dt,J=17.00, 1.70 Hz), 4.73 (1H, dt, J=11.00, 1.70 Hz), 3.85-3.81 (1H, m),3.51 (1H, ddd, J=8.42, 5.73, 2.07 Hz), 3.16 (1H, d, J=9.50 Hz), 3.05(1H, d, J=9.50 Hz), 2.85 (2H, dd, J=4.63, 2.20 Hz), 2.12-1.92 (2H, m),1.85 (1H, t, J=2.68 Hz).

(6) The compound A-22 (534.4 mg, 2.26 mmol) obtained in (5) wasdissolved in anhydrous pyridine (7.5 mL) and, after the addition ofpivaloyl chloride (0.276 mL, 2.26 mmol) at 0° C., the mixture wasstirred at the same temperature for 45 minutes. Saturated aqueous sodiumhydrogen carbonate was added to the mixture. The mixture was dilutedwith toluene and washed with brine. The organic layer was dried overanhydrous magnesium sulfate and evaporated to give the residue. Theresidue was diluted with dry dichloromethane (10 mL), and to thesolution were added 2,6-lutidine (1.1 mL, 9.22 mmol) andt-butyldimethylsilyl trifluoromethanesulfonate (1.7 mL, 7.55 mmol) at 0°C. The reaction mixture was stirred at same temperature for 1.5 hours.The reaction was diluted with ethyl acetate and the organic layer waswashed with brine, dried over anhydrous magnesium sulfate andevaporated. The residue was purified by silica gel column chromatography(hexane/ethyl acetate=99/1→85/15) to obtain A-23 (1.08 g, yield 91%).

¹H-NMR (CDCl₃) δ: 6.00-5.91 (1H, m), 5.21 (1H, d, J=17.32 Hz), 5.13 (1H,d, J=11.00 Hz), 4.32 (1H, dd, J=7.07, 3.90 Hz), 4.03 (2H, dd, J=19.03,11.22 Hz), 3.94 (1H, dd, J=10.73, 5.85 Hz), 3.64 (1H, d, J=9.76 Hz),3.45 (1H, d, J=9.76 Hz), 3.39 (1H, t, J=4.27 Hz), 2.51 (1H, ddd,J=16.83, 6.00, 3.00 Hz), 2.36 (1H, ddd, J=16.71, 6.10, 2.56 Hz), 1.95(1H, t, J=2.56 Hz), 1.19 (9H, s), 0.90 (9H, s), 0.88 (9H, s), 0.55-0.483H, m), 0.11 (3H, s), 0.09 (3H, s), 0.06 (3H, s), 0.03 (3H, s).

(7) Using the compound A-23 (70 mg, 0.15 mmol) obtained in (6) and thecompound B-3 (69 mg, 0.16 mmol) obtained in Example 1 (4) as startingmaterials, synthesis was carried out in the same manner as in Example 1(5) to obtain compound AB-3 (48.1 mg, 37.4%).

¹H-NMR (CDCl₃) δ: 6.18 (1H, d, J=10.98 Hz), 6.02 (1H, d, J=11.47 Hz),5.32 (1H, s), 5.01 (1H, s), 4.47 (1H, s), 4.03 (1H, q, J=4.15 Hz), 3.91(1H, d, J=9.03 Hz), 3.58 (1H, dd, J=11.10, 4.03 Hz), 3.46-3.39 (2H, m),3.32 (1H, d, J=9.51 Hz), 3.21 (1H, br s), 2.80 (1H, t, J=7.81 Hz), 2.61(1H, d, J=13.42 Hz), 2.24 (1H, dd, J=16.34, 3.42 Hz), 2.10 (1H, dd,J=13.66, 4.15 Hz), 2.05-1.84 (4H, m), 1.66-1.49 (12H, m), 1.43-1.30 (4H,m), 1.07 (4H, d, J=6.59 Hz), 0.98-0.83 (36H, m), 0.82-0.81 (2H, m),0.70-0.64 (9H, m), 0.57-0.54 (6H, m), 0.51-0.36 (6H, m), 0.11 (3H, s),0.10 (3H, s), 0.08 (3H, s), 0.07 (3H, s).

(8) Using the compound AB-3 (48.1 mg, 0.056 mmol) obtained in (7) as araw material, treatments were carried out in the same manner as inExample 1 (6). The reaction product (28.5 mg, 0.0327 mmol) was dissolvedin a mixed solvent of anhydrous dichloromethane/acetonitrile (1/1, 1 mL)and the solution was cooled to 0° C. Thereafter, tosylic acidmonohydrate (31 mg, 0.163 mmol) and lithium tetrafluoroborate (30 mg,0.327 mmol) were added, and the mixture was stirred at the sametemperature for 30 minutes. To the reaction mixture was added saturatedaqueous sodium hydrogen carbonate and the mixture was extracted withethyl acetate. The organic layer was dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The residue obtainedwas roughly purified by thin-layer silica gel chromatography (ethylacetate/acetone=9/1+0.5% acetic acid) and further purified byreversed-phase HPLC (A=95% water/acetonitrile; B=0.5% water/40%methanol/acetonitrile; B=85%) to obtain compound E-1 (4.9 mg, yield16.6%).

¹H-NMR (CDCl₃) δ: 6.41 (1H, d, J=11.22 Hz), 6.01 (1H, d, J=10.98 Hz),5.37 (1H, s), 5.08 (1H, d, J=1.46 Hz), 4.48 (1H, d, J=2.68 Hz),4.06-3.82 (2H, m), 3.55-3.25 (2H, m), 2.88-2.60 (2H, m), 2.28-1.54 (13H,m), 1.42-1.20 (10H, m), 1.10-1.08 (1H, m), 1.06 (3H, d, J=6.59 Hz), 0.91(3H, d, J=4.88 Hz), 0.54 (3H, s).

Example 10 Production of(5Z,7E)-(1R,2S,3R,20R)-2-(2-carboxyethoxy)-26,27-dimethyl-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (Compound F-1)

(1) Trimethylsilylacetylene (1.84 mL, 13.0 mmol) was dissolved in1,4-dioxane (15 mL) and, under an argon atmosphere and ice-bath cooling,thereto was added dropwise n-butyllithium (1.59 M n-hexane solution,8.18 mL, 13.0 mmol) over 10 minutes. Hereto was added compound B-4 (1.91g, 4.33 mmol) dissolved in 1,4-dioxane (10 mL), the compound B-4 beingsynthesized according to the method of Tanaka et al. (InternationalPublication No. WO 98/58909), and the mixture was heated under reflux at110° C. for 24 hours. After cooling to room temperature, saturatedaqueous ammonium chloride was added to the mixture, followed bystirring. Thereafter, the mixture was extracted with n-hexane, and theorganic layer obtained was washed with saturated aqueous sodiumchloride, dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The residue obtained was dissolved intetrahydrofuran-methanol (1:1, mL), potassium carbonate (718 mg, 5.20mmol) was added thereto, and the mixture was stirred at room temperatureovernight. Water was added to the reaction mixture and, thereafter, themixture was extracted with n-hexane. The organic layer obtained waswashed with saturated sodium chloride and dried over anhydrous sodiumsulfate. The organic layer was concentrated under reduced pressure andthe residue obtained was purified by silica gel column chromatography(n-hexane) to obtain compound B-5 (1.14 g, yield 89%).

¹H-NMR (CDCl₃) δ: 5.65 (1H, s), 2.90-2.86 (1H, m), 2.25 (1H, dt, J=16.6,3.0 Hz), 2.10-1.88 (5H, m), 1.72-1.25 (9H, m), 1.11 (3H, d, J=6.6 Hz),0.58 (3H, s).

(2) The compound B-5 (301 mg, 1.02 mmol) obtained in (1) was dissolvedin tetrahydrofuran (10 mL) and, under an argon atmosphere and cooling to−78° C., n-butyllithium (1.59 M n-hexane solution, 0.673 mL, 1.02 mmol)was added dropwise thereto, and the mixture was stirred for 30 minutes.Hereto was added 3-pentanone (0.216 mL, 2.04 mmol) and the mixture wasstirred for 1 hour with the temperature maintained at −78° C. To thereaction mixture was added saturated aqueous ammonium chloride and themixture was warmed to room temperature. The reaction mixture wasextracted with ethyl acetate, and the organic layer obtained was washedwith saturated aqueous sodium chloride and dried over anhydrous sodiumsulfate. The organic layer was concentrated under reduced pressure andthe residue obtained was purified by silica gel column chromatography(n-hexane/ethyl acetate=9/1) to obtain compound B-6 (205 mg, yield 53%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=11.2 Hz), 6.02 (1H, d, J=11.2 Hz), 5.39(1H, s), 5.10 (1H, s), 4.44 (1H, t, J=3.9 Hz), 4.11-4.07 (1H, m),3.84-3.81 (1H, m), 3.75-3.68 (2H, m), 3.39 (1H, dd, J=7.4, 3.3 Hz),2.84-2.81 (1H, m), 2.68 (1H, dd, J=13.7, 4.4 Hz), 2.52 (2H, t, J=6.8Hz), 2.29-2.20 (3H, m), 2.15-1.83 (6H, m), 1.70-1.22 (14H, m), 1.08-1.01(9H, m), 0.55 (3H, s) ppm.

(3) The compound B-6 (396 mg, 1.04 mmol) obtained in (2) was dissolvedin anhydrous N,N-dimethylformamide (4 mL), thereto were addedchlorotriethylsilane (0.283 mL, 1.68 mmol), imidazole (152 mg, 2.23mmol), and 4-dimethylaminopyridine (27 mg, 0.22 mmol), and the mixturewas stirred under heating at 50° C. for 1 hour. The reaction mixture wascooled to room temperature, anhydrous methanol (1 mL) was added thereto,and the mixture was stirred for 30 minutes. The mixture was diluted withtoluene and washed with saturated aqueous sodium chloride. Thereafter,the organic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue obtained was purifiedby silica gel column chromatography (n-hexane/ethyl acetate=90/10) toobtain compound B-7 (454.8 mg, yield 88%).

¹H-NMR (CDCl₃) δ: 5.65 (1H, s), 2.91-2.85 (1H, m), 2.24 (1H, dd,J=16.46, 3.54 Hz), 2.10 (1H, dd, J=16.58, 6.83 Hz), 2.02-1.88 (4H, m),1.71-1.58 (9H, m), 1.54-1.24 (7H, m), 1.08 (3H, d, J=8.00 Hz), 0.98-0.91(22H, m), 0.73-0.64 (9H, m), 0.58 (3H, s), 0.52 (2H, q, J=7.97 Hz).

(4) The compound A-4 (457 mg, 1 mmol) obtained in Example 2 (1) wasdissolved in anhydrous N,N-dimethylformamide (5 mL), thereto were addedtriethylamine (0.421 mL, 3 mmol) and chloromethyl benzyl ether (0.276mL, 2 mmol), and the mixture was stirred at 0° C. for 1 hour 45 minutesSaturated aqueous sodium hydrogen carbonate was added thereto and themixture was extracted with ethyl acetate. The organic layer was washedwith saturated aqueous sodium chloride, dried over anhydrous magnesiumsulfate, and, thereafter, concentrated under reduced pressure. Theresidue obtained was purified by silica gel column chromatography(n-hexane/ethyl acetate=95/5) to obtain compound A-24 (485 mg, yield84%).

(5) Using the compound B-7 (44 mg, 0.09 mmol) obtained in (3) and thecompound A-24 (43 mg, 0.075 mmol) obtained in (4) as raw materials, thecoupling reaction and the deprotection reaction were carried out in thesame manner as in Example 5 (4). The crude reaction product obtained wasroughly purified by thin-layer silica gel chromatography (ethylacetate/acetone=4/1+acetic acid (1.5 v/v %)) and further purified byreversed-phase HPLC (A=95% water/acetonitrile; B=0.5% water/40%methanol/acetonitrile; B=75%) to obtain compound F-1 (4.7 mg, yield12%).

¹H-NMR (CDCl₃) δ: 6.42 (1H, d, J=10.98 Hz), 6.00 (1H, d, J=10.98 Hz),5.37 (1H, d, J=1.46 Hz), 5.08 (1H, d, J=1.95 Hz), 4.47 (1H, d, J=2.93Hz), 4.08-3.94 (2H, m), 3.82-3.74 (1H, m), 3.33 (1H, dd, J=8.17, 3.05Hz), 2.83 (1H, d, J=12.20 Hz), 2.69-2.60 (3H, m), 2.30-2.20 (2H, m),1.98 (2H, d, J=11.71 Hz), 1.91-1.80 (1H, m), 1.72-1.24 (16H, m), 1.07(3H, d, J=6.34 Hz), 1.03 (8H, t, J=7.44 Hz), 0.54 (3H, s).

Example 11 Production of(5Z,7E)-(1R,2S,3R,20R)-2-(2-carboxyethoxy)-26,27-nor-25-cyclopentyl-23-yne-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol(Compound F-2) Formula 19

(1) Using the compound B-5 (442 mg, 1.5 mmol) obtained in Example 10 (1)as a starting material, synthesis was carried out in the same manner asin Example 10 (2) to obtain a mixture (427.2 mg) of compound B-8 andcyclopentanone. Using this crude material as a starting material andusing anhydrous N,N-dimethylformamide (4.5 mL), chlorotriethylsilane(0.283 mL, 1.68 mmol), imidazole (152 mg, 2.23 mmol), and4-dimethylaminopyridine (27 mg, 0.22 mmol), synthesis was carried out inthe same manner as in Example 10 (3) to obtain compound B-9 (506.2 mg,yield 68%).

¹H-NMR (CDCl₃) δ: 5.65 (1H, s), 2.92-2.85 (1H, m), 2.24 (1H, dd,J=16.46, 3.29 Hz), 2.08 (1H, dd, J=16.10, 6.83 Hz), 2.02-1.57 (19H, m),1.54-1.26 (7H, m), 1.07 (4H, d, J=7.56 Hz), 0.98-0.91 (15H, m),0.73-0.63 (8H, m), 0.57 (3H, s), 0.52 (3H, q, J=7.97 Hz).

(2) Using the compound B-9 (44 mg, 0.09 mmol) obtained in (1) and thecompound A-24 (43 mg, 0.075 mmol) obtained in Example 10 (4) as startingmaterials, synthesis was carried out in the same manner as in Example 10(5) to obtain compound F-2 (2.0 mg, yield 5%).

¹H-NMR (CDCl₃) δ: 6.41 (1H, d, J=10.98 Hz), 6.00 (1H, d, J=10.98 Hz),5.36 (1H, s), 5.07 (1H, s), 4.46 (1H, s), 4.10-3.93 (2H, m), 3.78 (1H,br s), 3.30 (1H, d, J=6.59 Hz), 3.07-2.62 (9H, m), 2.30-2.19 (2H, m),2.05-1.24 (25H, m), 1.06 (3H, d, J=6.59 Hz), 0.54 (3H, s).

Example 12 Evaluation of VDR Affinity

VDR was evaluated by using a commercial measurement and evaluation kit,for example, “PolarScreen Vitamin D Receptor Competitor Assay, Red, Cat.No. PV4569” marketed by Invitrogen Corporation, according to thefollowing procedure.

Solutions of the compounds were added to two wells each of a 384-wellblack plate in 10 μl aliquots. To each well, VDR/Fluoromone VDR Complexincluded in the kit was added in 10 μl aliquots and allowed to react atroom temperature for 2 hours. After 2 hours, fluorescence polarizationwas measured and VDR affinity was evaluated. In addition, the affinitywas evaluated in relative values (1/X) with the affinity of1,25-(OH)₂-vitamin D₃ taken as 1.

TABLE 2 Compound Name VDR Affinity (1/X) 1,25-(OH) 2-Vitamin D₃ 1/1Compound C-1 1/0.52 Compound D-1 1/0.92 Compound E-1 1/2.39 Compound F-11/1.23 Compound F-2 1/1.61

The compounds obtained according to the present invention were confirmedto have strong VDR affinity. Especially, Compound C-1 and Compound D-1were found to have very strong VDR affinity.

Example 13 VDR Transcriptional Activity in Human Osteoblast (HOS Cells)

(1) A reporter vector was constructed by inserting the sequence of thepromoter region of human ostocalcin gene into the upstream of luciferasegene using a pGL3 vector (Promega Corporation), wherein the promoterregion of the human ostocalcin gene was cloned using cDNA acquired fromHOS cells (purchased from ATCC) by a method known in the literature(Ozono et al., The Journal of Biological Chemistry, 265, 21881-21888(1990)). The expression vector was constructed by inserting a DNAsequence, which encodes human VDR and human RXR, into a pcDNA3 vector(Invitrogen Corporation). The HOS cells were incubated in a DMEM mediumcontaining 10% FBS under conditions of 37° C. and 5% CO₂, andsubcultured every 2 or 3 days.

(2) The cells which had been subcultured were recovered bycentrifugation and were suspended in serum- and phenol red-free DMEMmedium in a density of 4×10⁵ cells/mL. This was seeded on a 96-wellplate in an amount of 0.1 mL/well. To this system, various vectorsdescribed in (1) were added in an amount of 0.05 mL per well usingLipofectamine 2000 (Invitrogen Corporation). After incubation at 37° C.for 3 hours, 2 μl each of ethanol solutions of the test compounds ofvarious concentrations or ethanol as a control was added to each well.After incubation at 37° C. for 24 hours, the medium was removed, thecells were washed once with PBS(−), and thereafter luciferase activitywas measured by using a luminometer (Berthold Technologies GmbH & Co.KG) using Dual-Glo Luciferase Assay Kit (Promega Corporation).

As a result, all of the compounds of the present invention were found tohave transcriptional activity with EC₅₀ values of 20 nM or less.Further, Compounds C-1, C-2, D-1, E-1, F-1, and F-2 were found topossess transcriptional activity with EC₅₀ values of 0.2 nM or less.Especially, D-1, F-1, and F-2 were found to have transcriptionalactivity with EC₅₀ values of 0.02 nM or less.

Example 14 Bone Mineral Density-Enhancing Effect in Osteoporosis Model(Oophorectomy) Rats (Comparative Test)

Twelve-week old SD stock female rats (Charles River Japan, Inc.) weresubjected to bilateral oophorectomy and, after being left alone for 4weeks, the compounds of the present invention and2α-(3-hydroxypropyl)oxy-1α,25 -dihydroxyvitamin D₃ described inInternational Publication No. WO 01/62723 were each administered 5 timesa week for 4 weeks. After 24 hours from the final administration, bloodwas drawn under ether anesthesia and the rats were put down. Underanesthesia, bone mineral density of the fourth and fifth lumbarvertebrae was measured by using a dual-energy X-ray bone mineralanalyzer (QDR-2000; Hologic, Inc.). For comparison, a sham surgery(sham) group (abdominal operation is performed but the ovary is notremoved; the test compounds are not administered) and an oophorectomy(OVX) group (subjected to oophorectomy but the test compounds are notadministered) were also subjected to measurement of the bone mineraldensity of lumbar vertebrae at the time of dissection. Further,measurement of calcium concentration in the serum of each group was alsoperformed.

TABLE 3 Bone mineral Serum calcium Dose density value Group (ng/kg)(g/cm³) (mg/dL) Test 1 Sham — 0.2303 ± 0.0185  9.61 ± 0.16 OVX — 0.2048± 0.0139  9.64 ± 0.22 Compound C-1  4 0.2223 ± 0.0118 10.40 ± 1.00 100.2400 ± 0.0065 10.30 ± 0.20 Test 2 Sham — 0.2242 ± 0.0121 10.14 ± 0.17OVX — 0.2152 ± 0.0166  9.73 ± 0.15 Compound C-2  6 0.2196 ± 0.0177  9.83± 0.37 13 0.2308 ± 0.0081 10.19 ± 0.36 Test 3 Sham — 0.2338 ± 0.0120 9.71 ± 0.27 OVX — 0.2194 ± 0.0100  9.25 ± 0.11 Compound C-5 17 0.2316 ±0.0134  9.60 ± 0.15 50 0.2354 ± 0.0126 10.10 ± 0.21 Test 4 Sham — 0.2422± 0.0130 10.01 ± 0.04 OVX — 0.2163 ± 0.0100  9.58 ± 0.18 Compound C-7  60.2322 ± 0.0092  9.83 ± 0.22 13 0.2548 ± 0.0143 10.07 ± 0.22 Test 5 Sham— 0.2252 ± 0.0080  9.82 ± 0.22 OVX — 0.2115 ± 0.0110  9.53 ± 0.28Compound D-1  6 0.2211 ± 0.0175  9.75 ± 0.10 13 0.2360 ± 0.0143 10.23 ±0.26 Test 6 Sham — 0.2302 ± 0.0110  9.61 ± 0.16 OVX — 0.2042 ± 0.0070 9.64 ± 0.22 Compound D-6 50 0.2365 ± 0.0204  9.76 ± 0.29

TABLE 4 Bone mineral Serum calcium Dose density value Group (ng/kg)(g/cm³) (mg/dL) Test 7 Sham — 0.2252 ± 0.0080  9.82 ± 0.22 OVX — 0.2115± 0.0110  9.53 ± 0.28 Compound F-1  6 0.2366 ± 0.0139 10.12 ± 0.59Comparative Test Sham — 0.2451 ± 0.0251 10.50 ± 0.83 OVX — 0.2167 ±0.0126  9.60 ± 0.23 2α-(3- 10 0.2269 ± 0.0161 10.30 ± 0.37hydroxypropyl) 25 0.2473 ± 0.0157 11.20 ± 0.20 oxy-1α,25- dihydroxy-vitamin D₃

The bone mineral density of the OVX group was confirmed to decreasecompared to the sham surgery (sham) group by performing the operation.Also, the bone mineral density was confirmed to recover byadministration of vitamin D derivatives. However, the group which wasadministered with 2α-(3-hydroxypropyl)oxy-1α,25-dihydroxyvitamin D₃described in International Publication No. WO 01/62723 showed increasein the serum calcium value with increase in the bone mineral densityand, at a dose (25 ng/kg) necessary for the bone mineral density tobecome equal to or greater than the sham group, the serum calcium valuewas found to increase significantly, by 1 mg/dL or more. On the otherhand, the compounds of the present invention were found to enhance bonemineral density to a value equivalent to or greater than that of thesham group, while the increase in the serum calcium value compared tothat of the OVX group was found in a range of not more than 1 mg/dL.

From the results described above, the vitamin D₃ derivatives ormedicinally acceptable solvates thereof of the present invention werefound to have more excellent effects on bones than the heretoforereported vitamin D₃ derivatives.

The vitamin D₃ derivatives or medicinally acceptable solvates thereof ofthe present invention can be used as drugs.

1-13. (canceled)
 14. A compound represented by the following formula(1):

wherein R₂ represents a hydrogen atom or an alkyl group having 1 to 6carbon atoms or, together with the other R₂ and the carbon atom to whichthey are bound to, may form a cyclic alkyl group having 3 to 6 carbonatoms; R₄ represents a hydrogen atom, alkyl group having 1 to 6 carbonatoms, an alkylcarbonyloxyalkyl group with each alkyl having 1 to 6carbon atoms, an arylcarbonyloxyalkyl group with the aryl having 6 to 10carbon atoms and the alkyl having 1 to 6 carbon atoms, a methoxymethylgroup, a methoxyethoxymethyl group, a tetrahydrofuranyl group, atetrahydropyranyl group, or a benzyloxymethyl group; R₅ represents aprotecting group for a hydroxyl group; X represents an oxygen atom or amethylene group; and n represents an integer of 1 or 2.